Tfpi - 2 Interact With Prosaposin Research

Tissue factor pathway inhibitor-2 (TFPI-2), also named as placenta protein-5 (PP5) and matrix associated serine protease inhibitor (MSPI), is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor superfamily. The mature protein contains a short acidic amino-terminal region, three tandem Kunitz domains and a carboxyl-terminal tail highly enriched in basic amino acids. Several studies revealed that hTFPI-2 could inhibit several proteases activity including trypsin, plasmin, chymotrypsin, plasma kallikrein, cathepsin G and a wide variety of matrix metalloproteases(MMPs). Therefore, hTFPI-2 could inhibit invasive and metastatic ability of many tumors such as prostate cancer, lung cancer, fibrosarcoma, melanoma and gliomas, et al. Furthermore, the rupture of atherosclerosis plaque can also be inhibited by hTFPI-2. But the biological functions of TFPI-2 are still unclear. Especially, the interactions of TFPI-2 with other proteins haven’t been revealed well.We used the yeast two-hybrid approach to find TFPI-2-interacting proteins. These efforts resulted in the identification of several proteins as TFPI-2’s interaction partners, one of which is Prosaposin (PSAP). Studies have showed that PSAP is a multi-functional glycoprotein with versatile neurotrophic activities. It was identified initially as the precursor of sphingolipid activator proteins, saposins A-D, required for enzymatic hydrolysis of certain sphingolipids by lysosomal hydrolases. Mature saposins are generated from prosaposin through proteolytic processing to distribute to lysosomes, while PSAP is found as a secreted protein in various body fluids such as human milk, serum, cerebrospinal fluid and seminal plasma and also in neuronal and other plasma membranes.In this paper, the studies will focus on confirming the interaction between TFPI-2 and PSAP, as follow:1) With yeast two-hybrid screening, co-localization, GST-pull down and Co-immunoprecipitation experiments, we confirmed the interaction between TFPI-2 and PSAP.2) We obtained high purity and activity of recombination human PSAP-His proteins through Expression and purification of recombination proteins in HEK293T cells.3) Using two-hybrid assay, we found out KD2 of TFPI-2 contributing to interact with the C terminus of PSAP.4) Through cell invasion and immigration assays, we confirmed that PSAP can promote HT1080 cells invasion and migration and TFPI-2 can inhibit this process. Besides, gelatin zymography indicated that PSAP can enhance the activity of MMP-2 in HT1080 in a concentration-dependent manner and TFPI-2 can also block this enhancement.5) In addition, rhPSAP did not affect TFPI-2 function of inhibiting trypsin activity.6) We identified that TFPI-2 is gene silence in HT1080 and PSAP can be detected in HT1080 cells both at the mRNA and protein level. TFPI-2 did not effect the PSAP expression both at the mRNA or protein level in HT1080 cells.Conclusion:TFPI-2 can interact with the C terminus of PSAP through its second Kunitz domain and inhibit PSAP function of promoting HT1080 cells invasion and migration; PSAP has no influence on TFPI-2 function.